The regulated destruction of intracellular proteins is controlled by the ubiquitin-proteasome system (UPS) via tagging the ubiquitin on the proteins, and is essential to cellular protein homeostasis (1,2). The UPS has been extensively pursued as a drug target (3,4), with two proteasome inhibitors, Bortezomib and Carfilzomib, having been approved for the treatment of multiple myeloma (5-7).
The Cullin-Ring ligases (CRL), a central component of the UPS, regulate the turnover of approximately 20% of cellular proteins, and the dysregulation of CRLs plays a critical role in various human diseases, including cancer, cardiovascular diseases, neurodegenerative disorders, and viral infections (8-11). The activation of CRLs is controlled by NEDD8 (neural precursor cell expressed developmentally downregulated protein 8), a ubiquitin-like protein (9,10,12). Analogous to the process of ubiquitination, neddylation is a process by which the ubiquitin-like protein NEDD8 is conjugated to its target proteins.
The neddylation cascade begins with the activation of NEDD8 by an E1 enzyme, the NEDD8 activating enzyme (NAE), followed by transfer of the activated NEDD8 to one of two NEDD8-specific E2 enzymes, UBC12 and UBE2F. In the final step of this cascade, an E3 enzyme catalyzes the transfer of NEDD8 from E2 to target substrates (13). The enzymes of the NEDD8 pathway have been pursued as potential therapeutic targets (14-17) and MLN4924, an inhibitor of the E1 enzyme NAE, was shown to suppress tumor cell growth both in vitro and in vivo (18). Mechanistically, MLN4924 inhibits NAE enzymatic activity through formation of a covalent NEDD8-MLN4924 adduct, which in turn inactivates CRLs, leading to accumulation of CRL substrates (18,19). MLN4924 is currently being tested in clinical trials for the treatment of human cancers (20).
Schulman et al. have defined both the structural and biochemical mechanisms underlying the E1-E2-E3 cascade reaction in the NEDD8 pathway (13, 21-23). Schulman et al. further demonstrated that DCN1, a scaffold-like E3 ligase, facilitates the transfer of NEDD8 from UBC12 to cullins through its interaction with UBC12 and enhances the enzymatic activity of cullins (13,22,23). The co-crystal structure of the DCN1-UBC12 complex 22,23 reveals that UBC12 interacts with DCN1 through two distinct sites and the N-terminally acetylated UBC12 peptide binds to a well-defined pocket in DCN1.
To date, no small-molecule inhibitors of the DCN1-UBC12 interaction have been advanced into clinical development. Accordingly, a need still exists in the art for small molecule inhibitors of the UBC12-DCN1 protein-protein interaction, having physical and pharmacological properties that permit use of such inhibitors in a range of therapeutic applications in which modulation of the activity of cullins may have a therapeutic benefit.
Inhibitors of protein-protein interactions are generally considered to be difficult drugs to develop, because even when there is a well defined binding pocket on one of the proteins to target, that is rarely the totality of the mutual binding surface between the two entities. When inhibiting receptors or enzymes, there is often a small molecule ligand or cofactor which can be competed against, or a catalytic machinery which can be interfered with irrespective of substrate binding, and this allows relatively low affinity inhibitors to be potential drugs. However, with protein-protein interaction inhibitors (PPI inhibitors) it is frequently infeasible to block the whole of the interaction site between the two proteins, and if one is only blocking a part of the interaction site, very high affinity ligands are required in order to compete with the partner protein which will interact with a much larger protein surface than the inhibitor. Even with very good binding pockets, it is difficult to push binding affinities into the frequently required low picomolar range.
One answer to achieve highly potent inhibition of the protein-protein interaction is to use an inhibitor which forms a covalent bond to its target protein, as bond formation makes the effective binding between inhibitor and target protein much stronger. In recent years, this approach has been systematized, especially in the kinase inhibitor field, where a combination of intrinsically high affinity ligands, combined with a very precisely placed weak electrophile, usually close to a highly nucleophilic cysteine residue, has been shown to produce inhibitors which bind very strongly indeed to the target protein, but which are of intrinsically low enough chemical reactivity to have usable pharmacokinetics and acceptable off target toxicity profiles. For examples Afatinib, Ibrutinib and Osimertinib are all successful anticancer drugs which covalently attach to a cysteine on the edge of the ATP-binding domain in a small subset of kinases.
DCN1 has a cysteine (Cys115) on the edge of its deep UBC12 binding pocket, and in a suitable place whereby DCN1 inhibitors of the chemotype described in a previous patent application [U.S. Provisional Application No. 62/477,498], and illustrated herewithin, should be able to present a suitable electrophile in a manner to allow formation of a covalent bond between the cysteine sulfur atom and the abovementioned electrophile. Compounds of the present invention can bind to DCN1 as covalent inhibitors of the interaction between DCN1 and UBC12, and this leads to a major, highly consequential boost in their potency for these molecules as compared to their non-covalent inhibitor counterparts.